As part of the immune system, corneal epithelial cells have a crucial role in the defence against pathogens. The integrity of the cornea is endangered mainly by micro-organisms and UV-B irradiation. Cornea originated from mice and human was shown to express membrane bound Toll-Like Receptors (TLRs) that belong to the pattern recognition receptors (PRR) of the innate immunity and regulate the expression of several cytokines via NFkB pathway. Caterpiller molecules (Nalp/Nod) resemble to TLRs, but located in the cytosol and recognize intracellular pathogens or damaged self-molecules. They are capable of complex formation (e.g. inflammosome) and regulation of cytokine production. In preliminary studies using RT-PCR technique we have proven the constitutive expression of several Nalp/Nod molecules in human primary corneal epithelial cells, and inducible expression was proven in HaCaT and leukocyte cell lines. We plan to study the expression of Nalps/Nods in samples of human primary cells from healthy and pathological patients using Q-PCR and Western-blot techniques. We will study the inducible expression of these molecules upon treatment with TLR agonists and following UV irradiation in HCE-T cell line. With siRNA method we would like to study the regulation of NFkB pathway and the production of cytokines of the cell line in parallel. We aimed to find some explanation on the presence and the role of Caterpiller molecules in the functioning of the innate immunity of the eye.

In our work, we studied the expression and function of the intracellular pattern recognition receptor family, Nod-like receptors (NLRs) in human corneal epithelial cells and in keratinocytes. We found that (1) the expression of NLRs is decreased by UV-B irradiation; (2) the corneal epithelial cell line (HCE-T) fails to express the inflammasome adaptor ASC which results in the loss of IL-1beta expression in these cells; (3) the Nod2-signalosome, which participates in the responses against bacterial infections, functions properly in this cell line; (4) limbal stem cell (LSC) derived corneal epithelial cells express NLRs; (5) the expression level of NLRs from LSC is comparable with the NLR expression from photorefractive ceratectomy (PRK); (6) we established the Pseudomonas aeruginosa infection on LSCs to study the NLR function; (7) on human primary keratinocytes only the double stranded RNA-mimicking polyIC is able to induce the secretion of IL1-beta; (8) polyIC induces the expression of Nalp3 inflammosome components, induces caspase-1 enzymatic activity and caspase-1 inhibitor reduces IL-1beta production; (9) polyIC induces the expression of Nod2 and subsequent treatment with Nod2-specific agonist (MDP) synergistically induces the production of inflammatory cytokines (IL6, IL8, TNF) and antimicribial peptides (beta-defensin, CAMP).